Identification of a region of the bacteriophage T3 and T7 RNA polymerases that determines promoter specificity

Experimental interrogation of the path dependence and stochasticity of protein evolution using phage-assisted continuous evolution

“…To study the incidence of evolutionary convergence during protein evolution, we designed two selection pathways that subject T7 RNA polymerase (T7 RNAP) to selection pressure schedules with a common beginning and ending but that are otherwise distinct. Both pathways begin with phage encoding the wild-type T7 RNAP gene, which recognizes the T7 promoter with a high degree of sequence specificity (24), and diverge by demanding recognition of either the T3 or SP6 promoter, both of which are orthogonal to one another and to the T7 promoter in nature (25). Each pathway proceeds through a series of evolutionary "stepping stones" that introduce several nucleotide changes at a time ( Fig.…”

Identification of a minimal binding element within the T7 RNA polymerase promoter 1 1Edited by R. Ebright

“…Recent in vitro measurements of transcription from oligonucleotide-based promoters have identi®ed speci®c base functional groups involved in direct read-out of the sequence from position À11 to position À5, through contacts in the major groove (Li et al, 1996). Interactions with this upstream binding domain appear then to be typical of many duplex DNA binding proteins, and it is in this region that the primary sequence speci®city which distinguishes T7, T3, and SP6 RNA polymerases resides (Diaz et al, 1993;Joho et al, 1990;Raskin et al, 1992Raskin et al, ,1993. The fact that different species-distinguishing sequences are allowed in this region suggests that this region of the DNA is not directly involved in RNA polymerization and may serve simply as a binding tether for downstream elements.…”

T7 RNA polymerase mutants with altered promoter specificities.

“…In the case of T3 vs. T7 promoter specificity, the base pairs at -10 and -11 are the primary determinants of specific promoter recognition, and a T7 promoter variant having the corresponding T3 base pairs substituted at these positions (designated Pr7 -10C, -liC; the letter denotes the base on the nontemplate strand) is utilized by T3 RNAP but not by T7 RNAP (12). Previous work demonstrated that recognition of these base pairs involves the amino acid residue at position 748 (Asn) and that a T7 RNAP mutant having the corresponding T3 amino acid (Asp) at this position (denoted T7-N748D) preferentially utilizes PT7 -10C, -llC over a consensus T7 promoter (PrH) (10,13,14). To account for this specificity, it has been proposed that residue N748 makes specific hydrogen bonds with the base pair at -11 (and possibly at -10) in the major groove (10,11,14).…”